Process for reducing subvisible particles in a pharmaceutical formulation

ABSTRACT

The present disclosure provides a stable protein composition containing a surfactant and having less than 400 subvisible particles of 10 microns or greater diameter per container, or less than 10,000 subvisible particles of 2 microns or greater per container. A method of manufacturing such a stable protein composition is disclosed, which includes a unit of operation that removes or decreases an esterase activity that degrades the surfactant. The unit of operation may be hydrophobic interaction chromatography or filtration, mixed mode chromatography, or the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/061,797 filed Oct. 9, 2014, the disclosure of which is incorporatedherein by reference.

FIELD

This disclosure is directed to stable biopharmaceutical formulationcompositions that do not form meaningful amounts of subvisible particlesover time, and to methods of making the stable biopharmaceuticalformulations that do not form meaningful amounts of subvisible particlesover time. This disclosure is also directed to compositions and methodsof making biopharmaceutical formulations containing an intact emulsifieror stabilizer containing a fatty acid ester moiety.

BACKGROUND

Biopharmaceutical drugs (or biologics) often contain proteins or nucleicacid molecules. These molecules are quite often fairly large and proneto long term instability. In order to maintain efficacy over time,reduce immunogenicity and inflammatory reactions, and to meet regulatoryrequirements, these biologics must exhibit long term stability and acommercially reasonable shelf life.

Biological formulations may be subject to the formation of particulatematter over time during storage. Particles may be visible or subvisible.Subvisible particles are generally under 150 microns or 100 microns indiameter. Some particles may be “foreign”, that is a contaminant apartfrom the biological molecule. However, in those biological formulationscontaining proteins, the proteins may self-aggregate to form particles.Excessive handling, agitation, thermal stress, freeze-thaw, theintroduction of trace nanoparticles such as silicone oils and othersubstances associated with vials and syringe barrels may contribute toprotein aggregation and the formation of subvisible particles.Formulations having high protein concentrations, e.g., more concentratedthan about 30 mg/mL, are more prone to aggregation and subvisibleparticle formation.

Given the potential risks associated with the inclusion of subvisibleparticles, regulatory authorities like the FDA provide limitations onthe number of subvisible particles allowed in a pharmaceuticalformulation. For example, USP 31 monograph <788> sets the limit fornumber of particles allowed in parenteral formulations. For large volumeparenterals (greater than 100 mL), the limit is set at no more than 25particles of at least 10 microns per mL, and no more than 3 particles ofat least 25 microns per mL. For small volume parenterals (100 mL orless), the limit is set at no more than 6,000 particles of at least 10microns per container, and no more than 600 particles of at least 25microns per container.

To prevent or reduce the rate of particle formation in proteinaceousformulations, formulators of ordinary skill add stabilizers to theformulations. Those stabilizers include surface active agents andorganic co-solvents such as polysorbates surfactants and copolymers.Copolymers include for example ethylene oxide/polypropylene oxidecopolymers. Polysorbates generally used in pharmaceutical preparationsinclude polysorbate 20 and polysorbate 80, but others may be used aswell.

Polysorbates are fatty acid esters of PEG-ylated sorbitan(polyoxyethylene sorbitan esters). The polyoxyethylene serves as thehydrophilic head group and the fatty acid as the lipophilic tail. Theeffectiveness as a surfactant of the polysorbate depends upon bothgroups being present in a single molecule. When a polysorbate degrades(hydrolyzes) into its component head group and fatty acid tail, it losesits effectiveness as a protein stabilizer, potentially allowing foraggregation and subsequent subvisible particle formation. Therefore, inbiopharmaceutical formulations that employ polysorbates as proteinstabilizers, the stability of the polysorbates themselves is importantfor proper function and prevention of the formation of subvisibleparticles.

SUMMARY

The applicants have made the surprising discovery that an esterase,present as a host cell protein, may be co-purified with a protein ofinterest; and that the esterase may degrade the surfactant present in aformulation containing the protein of interest in some instances leadingto loss of surfactant activity and the formation of protein aggregatesand subvisible particles.

Thus, in one aspect the invention provides a composition comprising aprotein, a fatty acid ester surfactant, and less than 400 subvisibleparticles per container, wherein the subvisible particles have adiameter that is 10 microns or greater. In one embodiment, thecomposition comprises less than 10,000 subvisible particles, wherein thesubvisible particles have a diameter of at least 2 microns or more. Insome embodiments, the subvisible particles have a diameter of greaterthan or equal to ten microns. In others, the diameter of the subvisibleparticles is greater than or equal to 500 nanometers. In still otherembodiments, the diameter of the subvisible particles is greater than orequal to 25 microns. In some embodiments, the subvisible particles havea diameter that is less than 150 microns or less than 100 microns. Inother embodiments, the subvisible particles are greater than or equal to2 microns in diameter.

In one embodiment, the protein is an antibody. In some cases theconcentration of the antibody is high, for example at least 30 mg/mL, atleast 40 mg/mL, at least 80 mg/mL, or at least 100 mg/mL.

In one embodiment, the fatty acid ester comprises one or more fattyacids with an aliphatic tail of about 6 carbons to 21 carbons. In somecases the aliphatic tail has at least 18 carbons, such as oleate,linoleate, arachidonate, and the like. In other cases the aliphatic tailhas less than 18 carbons, such as laurate, caprate, caprylate,myristate, palmitate, palmitoleate, and the like. The fatty acid can besaturated, or unsaturated to various degrees. In some embodiments, thefatty acid ester is a detergent, such as an ionic, polar, or non-ionicdetergent.

In some embodiments, the fatty acid ester is a polyoxyethylene sorbitanfatty acid ester. In one embodiment, the fatty acid ester ispolyoxyethylene (20) sorbitan oleate. In another embodiment, the fattyacid ester is polyoxyethylene (20) sorbitan laurate.

In some embodiments, the composition has reduced levels of esteraseactivity such that the fatty acid ester in the composition remainsessentially intact. In some embodiments, the composition is free ofdetectable esterase activity. The esterase may be a carboxylic esterhydrolase (EC 3.1.1), such as for example a lipase. In one embodiment,the esterase is a phospholipase B-like 2 esterase, such as a Cricetulussp. phospholipase B-like 2 esterase. In a specific embodiment, theesterase comprises an amino acid sequence of SEQ ID NO:1.

In some embodiments, the composition comprises a buffer and/or a thermalstabilizer.

In some embodiments, the composition is or was stored for some time atsome temperature. In one embodiment, the composition was stored for atleast six months at 5° C.

In a second aspect, the invention provides a composition that contains aprotein and an intact fatty acid ester. What is meant by intact is thatthe fatty acid ester is essentially undegraded such that less than 20%by mole of the fatty acid ester is hydrolyzed into a free fatty acid andhead group. In other words, in a composition in which 20% of a fattyacid ester having a single fatty acid chain has hydrolyzed, there are 4moles of fatty acid ester per mole of free fatty acid. For fatty acidesters with two fatty acid chains, 20% hydrolyzed converts to 4 moles offatty acid ester per two moles of free fatty acid. For fatty acid esterswith three fatty acid chains, 20% hydrolyzed converts to 4 moles offatty acid ester per three moles of free fatty acid.

In one embodiment, no more than 15% of the fatty acid ester ishydrolyzed into the free fatty acid and head group (≧17 moles ofmonoester per 3 moles of free fatty acid), no more than 10% of the fattyacid ester is hydrolyzed into the free fatty acid and head group (≧9moles of monoester per mole of free fatty acid), or no more than 5% ofthe fatty acid ester is hydrolyzed into the free fatty acid and headgroup (≧19 moles of monoester per mole of free fatty acid).

In one embodiment, the protein is an antibody. In some cases theconcentration of the antibody is high, for example at least 30 mg/mL, atleast 40 mg/mL, at least 80 mg/mL, or at least 100 mg/mL.

In one embodiment, the fatty acid ester comprises one or more fattyacids with an aliphatic tail of from about 6 carbons to 21 carbons. Insome cases the aliphatic tail has at least 18 carbons, such as oleate,linoleate, arachidonate, and the like. In other cases the aliphatic tailhas less than 18 carbons, such as laurate, caprate, caprylate,myristate, palmitate, palmitoleate, and the like. The fatty acid can besaturated, or unsaturated to various degrees. In some embodiments, thefatty acid ester is a detergent, such as an ionic, a polar, or anon-ionic detergent.

In some embodiments, the fatty acid ester is a polyoxyethylene sorbitanfatty acid ester. In one embodiment, the fatty acid ester ispolyoxyethylene (20) sorbitan oleate. In another embodiment, the fattyacid ester is polyoxyethylene (20) sorbitan laurate.

In some embodiments, the composition has reduced levels of esteraseactivity such that the fatty acid ester in the composition remainsessentially intact. In some embodiments, the composition is free ofdetectable esterase activity. The esterase may be a carboxylic esterhydrolase (EC 3.1.1), such as for example a lipase. In one embodiment,the esterase is a phospholipase B-like 2 esterase, such as a Cricetulussp. phospholipase B-like 2 esterase. In a specific embodiment, theesterase comprises an amino acid sequence of SEQ ID NO:1.

In some embodiments, the composition comprises a buffer and/or a thermalstabilizer.

In some embodiments, the composition is or was stored for some time atsome temperature. In one embodiment, the composition was stored for atleast six months at 5° C.

In a third aspect, the invention provides a process for manufacturing astable protein formulation comprising the steps of (a) contacting ahydrophobic interaction media with a composition comprising a protein ofinterest and an esterase; and (b) collecting the protein of interestfrom the media. The protein of interest collected from the media (eitheras flow through in the loading or wash solution, or as elution in a washor elution solution) is associated with a reduced amount of esterase. Insome cases, no esterase activity is detected in the collectioncontaining the protein of interest.

In one embodiment, the protein of interest is an antibody, such as amonoclonal antibody, a bispecific antibody, and/or inter alia atherapeutic antibody, or fragments thereof.

In one embodiment, the esterase is a carboxylic ester hydrolase (EC3.1.1), such as for example a lipase. In one embodiment, the esterase iscapable of hydrolyzing a fatty acid ester of a sorbitan or iso-sorbide.In one embodiment, the esterase is a phospholipase B-like 2 esterase,such as a Cricetulus sp. phospholipase B-like 2 esterase or other rodentphospholipase B-like 2 esterase. In a specific embodiment, the esterasecomprises an amino acid sequence of SEQ ID NO:1.

In some embodiments, the collected protein of interest is furtherprocessed. In some cases, the collected protein of interest is subjectedto a buffer exchange, filtration, and/or additional chromatographicsteps. In one embodiment, the protein of interest is concentrated atsome point after collection.

In one embodiment, the collected protein of interest is combined at somepoint with a fatty acid ester that serves as a surfactant to preventprotein aggregation and/or subvisible particle formation. In some cases,the fatty acid ester is a polyoxyethylene (20) sorbitan ester, such aspolyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20)sorbitan monooleate.

In one embodiment, the protein of interest plus fatty acid estercombination is combined with a buffer and a thermal stabilizer.

In a fourth aspect, the invention provides a process for reducingesterase activity in a composition that contains a protein of interest,the process comprising the steps of (a) contacting a composition, whichcontains a protein of interest and an esterase, to a medium; (b)separating the protein of interest from the esterase; and (c) collectingthe protein of interest. A “medium” can be any format, such as forexample a chromatography resin, beads, cellulosic substrate, a membrane,or the like. In some embodiments the medium is a hydrophobic interactionmedium, which can be a hydrophobic interaction chromatography (HIC)resin or an HIC membrane. In other embodiments, the medium is a fattyacid affinity medium, which contains a ligand or adduct linked to asubstrate, such that the ligand or adduct binds to a fatty acid oraliphatic tail thereof.

In one embodiment, the protein of interest is an antibody, such as amonoclonal antibody, a bispecific antibody, and/or inter alia atherapeutic antibody, or fragments thereof.

In one embodiment, the esterase is a carboxylic ester hydrolase (EC3.1.1), such as for example a lipase. In one embodiment, the esterase iscapable of hydrolyzing a fatty acid ester of a sorbitan or iso-sorbide.In one embodiment, the esterase is a phospholipase B-like 2 esterase,such as a Cricetulus sp. phospholipase B-like 2 esterase or other rodentphospholipase B-like 2 esterase. In a specific embodiment, the esterasecomprises an amino acid sequence of SEQ ID NO: 1.

In some embodiments, the collected protein of interest is furtherprocessed. In some cases, the collected protein of interest is subjectedto a buffer exchange, filtration, and/or additional chromatographicsteps. In one embodiment, the protein of interest is concentrated atsome point after collection.

In one embodiment, the collected protein of interest is combined at somepoint with a fatty acid ester that serves as a surfactant to preventprotein aggregation and/or subvisible particle formation. In some cases,the fatty acid ester is a polyoxyethylene (20) sorbitan ester, such aspolyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20)sorbitan monooleate.

In one embodiment, the protein of interest plus fatty acid estercombination is combined with a buffer and a thermal stabilizer.

In a fifth aspect, the invention provides a process for reducing theformation of subvisible particles in a composition that contains aprotein of interest. The process comprises the steps of (a) removingesterase activity from the composition and (b) adding a fatty acid esterto the composition. In one embodiment, less than 400 particles having anaverage mean diameter of 10 microns or more form in a containercontaining the composition after storage for six months at 5° C. Inanother embodiment, less than 10,000 particles having an average meandiameter of 2 microns or more form in a container containing thecomposition after storage for six months at 5° C.

In one embodiment, the esterase is removed from the composition bycontacting the composition to a medium. The “medium” can be any format,such as chromatography resin, beads, a cellulosic substrate, a membrane,or the like. In some embodiments the medium is a hydrophobic interactionmedium, which can be a hydrophobic interaction chromatography (HIC)resin or an HIC membrane. In other embodiments, the medium is a fattyacid affinity medium, which contains a ligand or adduct linked to asubstrate, wherein the ligand or adduct binds to a fatty acid oraliphatic tail thereof.

In one embodiment, the protein of interest is an antibody, such as amonoclonal antibody, a bispecific antibody, and/or inter alia atherapeutic antibody, or fragments thereof.

In one embodiment, the esterase is a carboxylic ester hydrolase (EC3.1.1), such as for example a lipase. In one embodiment, the esterase iscapable of hydrolyzing a fatty acid ester of a sorbitan or aniso-sorbide. In a specific embodiment, the esterase preferentiallyhydrolyzes polyoxyethylene (20) sorbitan monolaurate overpolyoxyethylene (20) sorbitan monooleate. In one embodiment, theesterase is a phospholipase B-like 2 esterase, such as a Cricetulus sp.phospholipase B-like 2 esterase or other rodent phospholipase B-like 2esterase. In a specific embodiment, the esterase comprises an amino acidsequence of SEQ ID NO:1.

In some embodiments, the collected protein of interest is furtherprocessed. In some cases, the collected protein of interest is subjectedto a buffer exchange, filtration, and/or additional chromatographicsteps. In one embodiment, the protein of interest is concentrated atsome point after collection.

In one embodiment, the collected protein of interest is combined at somepoint with a fatty acid ester that serves as a surfactant or emulsifierto prevent protein aggregation and/or subvisible particle formation. Insome cases, the fatty acid ester is a polyoxyethylene (20) sorbitanester, such as polyoxyethylene (20) sorbitan monolaurate orpolyoxyethylene (20) sorbitan monooleate.

In one embodiment, the protein of interest plus fatty acid estercombination is combined with a buffer and a thermal stabilizer.

In one embodiment, the composition is stored at 5° C. for at least 6months.

DRAWINGS

FIG. 1 depicts a chromatogram showing relative amounts of differentmolecular species within (A) lower quality polysorbate 20 (PS20-A), (B)higher quality polysorbate 20 (PS20-B), and (C) polysorbate 80 (PS80).

FIG. 2 depicts a graph showing the correlation between phospholipaseactivity (in parts per million) and percent degradation of polysorbate20.

DETAILED DESCRIPTION

This invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention is defined bythe claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned are herebyincorporated by reference.

DEFINITIONS

The term “protein” means any amino acid polymer having more than about20 amino acids covalently linked via amide bonds. Proteins contain oneor more amino acid polymer chains, generally known in the art as“polypeptides”. Thus, a polypeptide may be a protein, and a protein maycontain multiple polypeptides to form a single functioning biomoleculeof a single conformation. Disulfide bridges (between cysteine residuesto form cystine) may be present in some proteins. These covalent linksmay be within a single polypeptide chain, or between two individualpolypeptide chains. For example, disulfide bridges are essential toproper structure and function of insulin, immunoglobulins, protamine,and the like. For a recent review of disulfide bond formation, see Okaand Bulleid, “Forming disulfides in the endoplasmic reticulum,” BiochimBiophys Acta, 2013 November; 1833(11):2425-9.

In addition to disulfide bond formation, proteins may be subject toother post-translational modifications. Those modifications includelipidation (e.g., myristoylation, palmitoylation, farnesoylation,geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchorformation), alkylation (e.g., methylation), acylation, amidation,glycosylation (e.g., addition of glycosyl groups at arginine,asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, and/ortryptophan), and phosphorylation (i.e., the addition of a phosphategroup to serine, threonine, tyrosine, and/or histidine). For a recentreview on the post-translational modification of proteins produced ineukaryotes, see Mowen and David, “Unconventional post-translationalmodifications in immunological signaling,” Nat Immunol, 2014 June;15(6):512-20; and Blixt and Westerlind, “Arraying the post-translationalglycoproteome (PTG),” Curr Opin Chem Biol, 2014 February; 18:62-9.

Immunoglobulins are proteins having multiple polypeptide chains andextensive post-translational modifications. The canonical immunoglobulinprotein (e.g., IgG) comprises four polypeptide chains—two light chainsand two heavy chains. Each light chain is linked to one heavy chain viaa cystine disulfide bond, and the two heavy chains are bound to eachother via two cystine disulfide bonds. Immunoglobulins produced inmammalian systems are also glycosylated at various residues (e.g., atasparagine residues) with various polysaccharides, and can differ fromspecies to species, which may affect antigenicity for therapeuticantibodies (see Butler and Spearman, “The choice of mammalian cell hostand possibilities for glycosylation engineering”, Curr Opin Biotech,2014 December; 30:107-112).

As used herein, “protein” includes biotherapeutic proteins, recombinantproteins used in research or therapy, trap proteins and other Fc-fusionproteins, chimeric proteins, antibodies, monoclonal antibodies, humanantibodies, bispecific antibodies, antibody fragments, nanobodies,recombinant antibody chimeras, cytokines, chemokines, peptide hormones,and the like. Proteins may be produced using recombinant cell-basedproduction systems, such as the insect bacculovirus system, yeastsystems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHOderivatives like CHO-K1 cells). For a recent review discussingbiotherapeutic proteins and their production, see Ghaderi et al.,“Production platforms for biotherapeutic glycoproteins. Occurrence,impact, and challenges of non-human sialylation,” Biotechnol Genet EngRev. 2012; 28:147-75.

The term “antibody”, as used herein, includes immunoglobulin moleculescomprised of four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chaincomprises a heavy chain variable region (abbreviated herein as HCVR orVH) and a heavy chain constant region. The heavy chain constant regioncomprises three domains, CH1, CH2 and CH3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region comprisesone domain, CL. The VH and VL regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each VH and VL is composed of three CDRs andfour FRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRsmay be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may beabbreviated as LCDR1, LCDR2 and LCDR3. The term “high affinity” antibodyrefers to those antibodies having a binding affinity to their target ofat least 10-9 M, at least 10-1 M; at least 10-11 M; or at least 10-12 M,as measured by surface plasmon resonance, e.g., BIACORE™ orsolution-affinity ELISA.

The phrase “bispecific antibody” includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodiesgenerally comprise two different heavy chains, with each heavy chainspecifically binding a different epitope—either on two differentmolecules (e.g., antigens) or on the same molecule (e.g., on the sameantigen). If a bispecific antibody is capable of selectively binding twodifferent epitopes (a first epitope and a second epitope), the affinityof the first heavy chain for the first epitope will generally be atleast one to two or three or four orders of magnitude lower than theaffinity of the first heavy chain for the second epitope, and viceversa. The epitopes recognized by the bispecific antibody can be on thesame or a different target (e.g., on the same or a different protein).Bispecific antibodies can be made, for example, by combining heavychains that recognize different epitopes of the same antigen. Forexample, nucleic acid sequences encoding heavy chain variable sequencesthat recognize different epitopes of the same antigen can be fused tonucleic acid sequences encoding different heavy chain constant regions,and such sequences can be expressed in a cell that expresses animmunoglobulin light chain. A typical bispecific antibody has two heavychains each having three heavy chain CDRs, followed by (N-terminal toC-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, andan immunoglobulin light chain that either does not conferantigen-binding specificity but that can associate with each heavychain, or that can associate with each heavy chain and that can bind oneor more of the epitopes bound by the heavy chain antigen-bindingregions, or that can associate with each heavy chain and enable bindingor one or both of the heavy chains to one or both epitopes.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain constant region sequence from any organism,and unless otherwise specified includes a heavy chain variable domain.Heavy chain variable domains include three heavy chain CDRs and four FRregions, unless otherwise specified. Fragments of heavy chains includeCDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has,following the variable domain (from N-terminal to C-terminal), a CH1domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragmentof a heavy chain includes a fragment that is capable of specificallyrecognizing an antigen (e.g., recognizing the antigen with a KD in themicromolar, nanomolar, or picomolar range), that is capable ofexpressing and secreting from a cell, and that comprises at least oneCDR.

The phrase “light chain” includes an immunoglobulin light chain constantregion sequence from any organism, and unless otherwise specifiedincludes human kappa and lambda light chains. Light chain variable (VL)domains typically include three light chain CDRs and four framework (FR)regions, unless otherwise specified. Generally, a full-length lightchain includes, from amino terminus to carboxyl terminus, a VL domainthat includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constantdomain. Light chains that can be used with this invention include those,e.g., that do not selectively bind either the first or second antigenselectively bound by the antigen-binding protein. Suitable light chainsinclude those that can be identified by screening for the most commonlyemployed light chains in existing antibody libraries (wet libraries orin silico), where the light chains do not substantially interfere withthe affinity and/or selectivity of the antigen-binding domains of theantigen-binding proteins. Suitable light chains include those that canbind one or both epitopes that are bound by the antigen-binding regionsof the antigen-binding protein.

The phrase “variable domain” includes an amino acid sequence of animmunoglobulin light or heavy chain (modified as desired) that comprisesthe following amino acid regions, in sequence from N-terminal toC-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. A “variable domain” includes an amino acid sequence capableof folding into a canonical domain (VH or VL) having a dual beta sheetstructure wherein the beta sheets are connected by a disulfide bondbetween a residue of a first beta sheet and a second beta sheet.

The phrase “complementarity determining region,” or the term “CDR,”includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild-typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule (e.g., an antibodyor a T cell receptor). A CDR can be encoded by, for example, a germlinesequence or a rearranged or unrearranged sequence, and, for example, bya naive or a mature B cell or a T cell. In some circumstances (e.g., fora CDR3), CDRs can be encoded by two or more sequences (e.g., germlinesequences) that are not contiguous (e.g., in an unrearranged nucleicacid sequence) but are contiguous in a B cell nucleic acid sequence,e.g., as the result of splicing or connecting the sequences (e.g., V-D-Jrecombination to form a heavy chain CDR3).

The phrase “Fc-containing protein” includes antibodies, bispecificantibodies, immunoadhesins, and other binding proteins that comprise atleast a functional portion of an immunoglobulin CH2 and CH3 region. A“functional portion” refers to a CH2 and CH3 region that can bind a Fcreceptor (e.g., an FcγR; or an FcRn, i.e., a neonatal Fc receptor),and/or that can participate in the activation of complement. If the CH2and CH3 region contains deletions, substitutions, and/or insertions orother modifications that render it unable to bind any Fc receptor andalso unable to activate complement, the CH2 and CH3 region is notfunctional.

Fc-containing proteins can comprise modifications in immunoglobulindomains, including where the modifications affect one or more effectorfunction of the binding protein (e.g., modifications that affect FcγRbinding, FcRn binding and thus half-life, and/or CDC activity). Suchmodifications include, but are not limited to, the followingmodifications and combinations thereof, with reference to EU numberingof an immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254,255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307,308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359,360, 361, 362, 373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389,398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

For example, and not by way of limitation, the binding protein is anFc-containing protein and exhibits enhanced serum half-life (as comparedwith the same Fc-containing protein without the recited modification(s))and have a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at 428 and/or 433 (e.g.,L/R/SI/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification at 250and/or 428; or a modification at 307 or 308 (e.g., 308F, V308F), and434. In another example, the modification can comprise a 428L (e.g.,M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I),and a 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434(e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and256E) modification; a 250Q and 428L modification (e.g., T250Q andM428L); a 307 and/or 308 modification (e.g., 308F or 308P).

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In some embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In some embodiments,the cell is eukaryotic and is selected from the following cells: CHO(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell,Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK),HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21),Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myelomacell, tumor cell, and a cell line derived from an aforementioned cell.In some embodiments, the cell comprises one or more viral genes, e.g. aretinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

The term “fatty acid ester” means any organic compound that contains afatty acid chain linked to a head group via an ester bond. An ester bondis formed when a hydroxyl group (e.g., an alcohol or carboxylic acid) isreplaced by an alkoxy group. As used herein, the hydroxyl group can bepart of a carboxylic acid, more specifically a fatty acid, and/or analcohol, such as glycerol, sorbitol, sorbitan, isosorbide, or the like.The alcohol group is generally referred to herein as the head group.

Examples of fatty acid esters generally include phospholipids, lipids(e.g., the head group is glycerol, including monoglycerides,diglycerides, and triglycerides), and surfactants and emulsifiers,including for example polysorbates like polysorbate 20, polysorbate 60,and polysorbate 80, which are non-ionic detergents. Surfactants andemulsifiers are useful as cosolvents and stabilizers. They function byassociating with both a hydrophilic surface and a lipophilic surface tomaintain dispersion and structural stability of ingredients, likeproteins. Surfactants are added to protein formulations primarily toenhance protein stability against mechanical stress, such as air/liquidinterface and solid/liquid interface shear. Without a surfactant,proteins may in some cases become structurally unstable in solution, andform multimeric aggregates that eventually become subvisible particles.

The term “fatty acid” or “fatty acid chain” means a carboxylic acidhaving an aliphatic tail. An aliphatic tail is simply a hydrocarbonchain comprising carbon and hydrogen, and in some cases, oxygen, sulfur,nitrogen and/or chlorine substitutions. Aliphatic tails can be saturated(as in saturated fatty acids), which means that all carbon-carbon bondsare single bonds (i.e., alkanes). Aliphatic tails can be unsaturated (asin unsaturated fatty acids), wherein one or more carbon-carbon bonds aredouble bonds (alkenes), or triple bonds (alkanes).

Fatty acids are generally designated as short-chain fatty acids, whichhave fewer than six carbons in their aliphatic tails, medium-chain fattyacids having six to twelve carbons, long-chain fatty acids havingthirteen to twenty one carbons, and very long chain fatty acids havingaliphatic tails of twenty two carbons and longer. As mentioned above,fatty acids are also categorized according to their degree ofsaturation, which correlates to stiffness and melting point. Commonfatty acids include caprylic acid (8 carbons:0 double bonds; 8:0),capric acid (10:0), lauric acid (12:0), myristic acid (14:0),myristoleic acid (14:1), palmitic acid (16:0), palmitoleic acid (16:1),sapienic acid (16:1), stearic acid (18:0), oleic acid (18:1), elaidicacid (18:1), vaccenic acid (18:1), linoleic acid (18:2), linelaedic acid(18:2), alpha-linolenic acid (18:3), arachidic acid (20:0), arachidonicacid (20:4), eicosapentaenoic acid (20:5), behenic acid (22:0), erucicacid (22:1), docosahexaenoic acid (22:6), lignoceric acid (24:0), andcerotic acid (26:0).

As mentioned above, polysorbates are fatty acid esters useful asnon-ionic surfactants and protein stabilizers. Polysorbate 20,polysorbate 40, polysorbate 60, and polysorbate 80 are widely employedin the pharmaceutical, cosmetic, and food industries as stabilizers andemulsifiers. Polysorbate 20 mostly comprises the monolaurate ester ofpolyoxyethylene (20) sorbitan. Polysorbate 40 mostly comprises themonopalmitate ester of polyoxyethylene (20) sorbitan. Polysorbate 60mostly comprises the monostearate ester of polyoxyethylene (20)sorbitan. Polysorbate 80 mostly comprises the monooleate ester ofpolyoxyethylene (20) sorbitan.

The quality of commercial grades of polysorbates varies from vendor tovendor. Polysorbates therefore are often mixtures of various chemicalentities, consisting mostly of polyoxyethylene (20) sorbitan monoesters(as described above) with, in some cases, isosorbide ester contaminants.The head group (in this case polyoxyethylene (20) sorbitan) comprises asorbitan (a mixture of dehydrated sorbitols, including1,4-anhydrosorbitol, 1,5-anhydrosorbitol, and 1,4,3,6-dianhydrosorbitol)substituted at three of its alcohol groups to form ether bonds withthree polyoxyethylene groups. The fourth alcohol group is substitutedwith a fatty acid to form a fatty acid ester.

In some commercially available batches of polysorbates, the polysorbatecontains isosorbide monoesters. Isosorbide is a heterocyclic derivativeof glucose, also prepared by the dehydration of sorbitol. It is a diol,i.e., having two alcohol groups that can take part in the formation ofone or two ester bonds. Thus, for example, some lots of polysorbate 20can contain significant amounts of isosorbide laurate mono- anddi-esters.

In addition to head group variation, preparations of polysorbatescontain variable amounts of other fatty acid esters. For example, ananalysis of one particular source of polysorbate 20 revealed <10%caprylic acid, <10% capric acid, 40-60% lauric acid, 14-25% myristicacid, 7-15% palmitic acid, <11% oleic acid, <7% stearic acid, and <3%linoleic acid. An analysis of a polysorbate 80 batch revealed <5%myristic acid, <16% palmitic acid, >58% oleic acid, <6% stearic acid,and <18% linoleic acid.

Biopharmaceutical drugs are often formulated with non-ionic detergentslike polysorbate 20 or polysorbate 80. These detergents help stabilizelarge molecules like antibodies and other proteins, and help prevent theformation of supermolecular ternary complexes or other aggregates.Aggregates can become nanoparticles or subvisible particles in the 10 to100 micron range or 2 to 100 micron range, and interfere with drugproduct stability and shelf-life. Therefore, the stability of proteinformulations depends in some cases upon the stability of the non-ionicdetergent additive.

The phrase “subvisible particle” means a particle that is not visible,especially in a liquid. In other words, a solution or other liquidcontaining subvisible particles, but not visible particles, will notappear cloudy. Subvisible particles generally include those particles100 micron or less in diameter, but in some cases include particlesunder 150 microns (Narhi et al., “A critical review of analyticalmethods for subvisible and visible particles,” Curr Pharm Biotechnol10(4):373-381 (2009)). Subvisible particles may be the result of foreigncontaminants or protein aggregation. Protein aggregates can be soft andamorphous in shape and therefore may be difficult to detect usingconventional light obscuration and microscopic methods (Singh and Toler,“Monitoring of subvisible particles in therapeutic proteins,” MethodsMol Biol. 2012; 899:379-401). Subvisible particles may comprise interalia silicone contaminants (oily droplets), free fatty acids (oilydroplets), aggregated protein (amorphous particles), and/orprotein/fatty acid complexes (amorphous particles).

Subvisible particles can be detected by any one or more of variousmethods. The USP standards specify light obscuration (Method 1) andoptical microscopy (Method 2) protocols. Other methods include flowimage analysis, Coulter counting, and submicron particle trackingmethods. For light obscuration (LO), particles are counted based on theshadows they cast upon a light detector as they pass through a lightbeam in a flow cell. The size, shape and inverse intensity of the shadowdepends upon the size, shape and difference in the refractive index ofthe particle relative to the solution. The lower size range fordetection using LO is about 2 microns. A commonly used LO device is theHIAC instrument (Beckman Coulter, Brea, Calif.).

Light obscuration is criticized for underestimating protein aggregatesand other amorphous structures. Flow image analysis, such as micro-flowimaging (MFI) (Brightwell Technologies, Ottawa, Ontario), is a moresensitive method of detecting the irregularly shaped, fragile, andtransparent proteinaceous subvisible particles, and of differentiatingthose types of particles from silicone micro-droplets, air bubbles, andother foreign contaminants (Sharma et al., “Micro-flow imaging: Flowmicroscopy applied to sub-visible particulate analysis in proteinformulations,” AAPS J. 12(3): 455-464 (2010)). Briefly, MFI is flowmicroscopy in which successive bright field images are taken andanalyzed in real time. Image analysis algorithms are applied to theimages to discriminate air bubbles, silicone oil droplets, andproteinaceous aggregates. Volumes as low as about 250 microliters to ashigh as tens of milliliters can be analyzed. Depending on the systemused, particles in the range of two to 300 microns, or one to 70 micronscan be detected (Id).

The FDA and other government regulatory agencies have placed limits onthe amount of subvisible particles allowed in parenteral drugformulations. The major articulated concern is the uncertaintysurrounding potential immunogenicity and downstream negative effects inthe patient receiving the drug (Singh et al., “An industry perspectiveon the monitoring of subvisible particles as a quality attribute forprotein therapeutics,” J. Pharma. Sci. 99(8):3302-21 (2010)). For smallvolume parenteral drugs (i.e., 25 mL or below), the pharmacopeia limitssubvisible particles (SVP) of greater than or equal to 10 microns to nomore than 6,000 SVPs per container, and SVPs of greater than or equal to25 microns to no more than 600 per container. (United StatesPharmacopeia and National Formulary (USP 33-NF 28), <788> ParticulateMatter in Injections.) For ophthalmic drugs, the SVP limits are 50 permL of 10 microns or greater, 5 per mL of 25 microns or greater, and 2per mL of 50 microns or greater (Id at <789> Particulate Matter inOphthalmic Solutions). Regulatory agencies are increasinglycontemplating that manufacturers establish specifications for SVPs of 2microns or greater (see Singh et al., “An industry perspective on themonitoring of subvisible particles as a quality attribute for proteintherapeutics,” J. Pharm. Sci. 99(8):3302-21 (2010)).

The term “esterase” means an enzyme that catalyzes the hydrolysis of anester bond to create an acid and an alcohol. Esterases are a diversecategory of enzymes, including acetyl esterases (e.g.,acetylcholinesterase), phosphatases, nucleases, thiolesterases, lipasesand other carboxyl ester hydrolases (EC 3.1. As its name implies acarboxyl ester hydrolase (a.k.a. carboxylesterase, carboxylic-esterhydrolase, and EC 3.1.1.1) uses water to hydrolyze a carboxylic esterinto an alcohol and a carboxylate. A lipase is a carboxyl esterhydrolase that catalyzes the hydrolysis of lipids, includingtriglycerides, fats and oils into fatty acids and an alcohol head group.For example, triglycerides are hydrolyzed by lipases like pancreaticlipase to form monoacylglycerol and two fatty acid chains.

Phospholipases are lipases that hydrolyze phospholipids into fatty acidsand other products. Phospholipases fall into four broad categories:phospholipase A (including phospholipase A1 and phospholipase A2),phospholipase B, and the phosphodiesterases phosphodiesterase C andphosphodiesterase D. In addition to the canonical phospholipases,phospholipase B-like enzymes, which reside at the lysosome lumen, arethought to be involved in lipid catalysis. For example, phospholipaseB-like 2 (PLBL2) is postulated to have esterase activity based uponsequence homology and subcellular localization (Jensen et al.,“Biochemical characterization and liposomal localization localization ofthe mannose-6-phosphate protein p′76,” Biochem. J. 402: 449-458 (2007)).

Applicants have discovered an enzymatic activity associated with thedestabilization of polysorbates (including polysorbate 20 andpolysorbate 80). That activity was found to be associated with anesterase, such as a polypeptide comprising the amino acid sequences ofTable 1. A BLAST search of those peptide sequences revealed identitywith a putative phospholipase B-like 2 (PLBL2). PLBL2 is highlyconserved in hamster, rat, mice, human and bovine. The applicantsenvision that PLBL2, which copurifies under certain processes with someclasses of proteins-of-interest manufactured in a mammalian cell line,has esterase activity responsible for the hydrolysis of polysorbate 20and 80. Applicants envision that other esterase species, of which PLBL2is an example, may contribute to polysorbate instability, depending uponthe particular protein-of-interest and/or genetic/epigenetic backgroundof the host cell.

Ester hydrolysis of polysorbate 80 was recently reported (see Labrenz,S. R., “Ester hydrolysis of polysorbate 80 in mAb drug product: evidencein support of the hypothesized risk after observation of visibleparticulate in mAb formulations,” J. Pharma. Sci. 103(8):2268-77(2014)). That paper reported the formation of visible particles in aformulation containing IgG. The author postulated that the colloidal IgGparticles formed due to the enzymatic hydrolysis of oleate esters ofpolysorbate 80. Although no esterase was directly identified, the authorspeculates that a lipase or tweenase copurified with the IgG, which wasresponsible for degrading the polysorbate 80. Interestingly, IgGsformulated with polysorbate 20 did not form particles and the putativeesterase did not hydrolyze the polysorbate 20. The author reported thatthe putative lipase associated with the IgG did not affect saturated C12fatty acid (i.e., laurate) (Id at 7.)

TABLE 1 Sequence Sequence Identifier Amino acid Sequence IdentifierAmino acid Sequence SEQ ID NO: 1 DLLVAHNTWNSYQNMLR SEQ ID NO: 16LTLLQLKGLEDSYEGR SEQ ID NO: 2 LIRYNNFLHDPLSLCEACIPKP SEQ ID NO: 17MSMLAASGPTWDQLPPFQ SEQ ID NO: 3 SVLLDAASGQLR SEQ ID NO: 18 VTSFSLAKRSEQ ID NO: 4 DQSLVEDMNSMVR SEQ ID NO: 19 QNLDPPVSR SEQ ID NO: 5QFNSGTYNNQWMIVDYK SEQ ID NO: 20 IIKKYQLQFR SEQ ID NO: 6QGPQEAYPLIAGNNLVFSSY SEQ ID NO: 21 AQIFQRDQSLVEDMNSMVR SEQ ID NO: 7SMLHMGQPDLWTFSPISVP SEQ ID NO: 22 LIRYNNFLHDPLSLCEACIPKP SEQ ID NO: 8YNNFLHDPLSLCEACIPKPNA SEQ ID NO: 23 SVLLDAASGQLR SEQ ID NO: 9LALDGATWADIFK SEQ ID NO: 24 DQSLVEDMNSMVR SEQ ID NO: 10 LSLGSGSCSAIIKSEQ ID NO: 25 DLLVAHNTWNSYQNMLR SEQ ID NO: 11 YVQPQGCVLEWIRSEQ ID NO: 26 YNNFLHDPLSLCEACIPKPNA SEQ ID NO: 12 RMSMLAASGPTWDQLPPFQSEQ ID NO: 27 RMSMLAASGPTWDQLPPFQ SEQ ID NO: 13 SFLEINLEWMQRSEQ ID NO: 28 SMLHMGQPDLWTFSPISVP SEQ ID NO: 14 VLTILEQIPGMVVVADADKTEDSEQ ID NO: 29 MSMLAASGPTWDQLPPFQ SEQ ID NO: 15 VRSVLLDAASGQLRSEQ ID NO: 30 VRSVLLDAASGQLR SEQ ID NO: 31 QNLDPPVSR

As used herein, the phrase “percent fatty ester hydrolysis” means themolar proportion of fatty acid ester that has been hydrolyzed. Sincehydrolysis of a fatty acid ester results in the release of a free fattyacid, the percent fatty ester hydrolysis can be determined by measuringthe free fatty acid in a sample. Therefore, percent fatty esterhydrolysis may be determined by calculating moles of free fatty aciddivided by the sum of moles of fatty acid plus moles of fatty acidester. In the case of percent hydrolysis of polysorbate 80 orpolysorbate 20, that number may be determined by calculating the molesof free oleate or laurate (i.e., free fatty acid, a.k.a. FFA) anddividing by the total moles of remaining polysorbate plus moles of freefatty acid.

The term “esterase inhibitor” means any chemical entity that reduces,inhibits, or blocks the activity of an esterase. The applicants envisionthat the inclusion of an esterase inhibitor in a protein formulationcontaining a fatty acid ester surfactant may help maintain proteinstability and help reduce SVP formation. Common esterases known in theart include orlistat (tetrahydrolipistatin; an inhibitor ofcarboxylesterase 2 and lipoprotein lipase), diethylumbelliferylphosphate (a cholesterol esterase [lipsase A] inhibitor), URB602([1-1′-biphenyl]-3-tl-carbamicacid cyclohexyl ester; a monoacylglycerollipase inhibitor), and 2-butoxyphenylboronic acid (an inhibitor ofhormone-sensitive lipase). The inclusion of an esterase inhibitor duringpurification of a protein of interest or in the final formulation isexpected to prevent or slow the hydrolysis of non-ionic detergents likepolysorbate 20 and polysorbate 80, which in turn is expected to preventor reduce subvisible particle formation.

The term “buffer” means a buffering solution or a buffering agent thatstabilizes the pH of a solution. A buffer generally comprises a weakacid and its conjugate base, or a weak base and its conjugate acid.Buffering of a protein solution at or close to the optimal pH helps toensure proper protein folding and function. The best buffer can beidentified for example by measuring the circular dichroism of theprotein (e.g., antibody) solution at various pHs. Circular dichrosim(CD) is one method used to determine structural changes (unfolding) of aprotein (S. Beychok, “Circular dichroism of biological macromolecules,”Science 154(3754):1288-99 (1966); Kemmer and Keller, “Nonlinearleast-squares data fitting in Excel spreadsheets,” Nat Protoc.5(2):267-81 (2010)). Some proteins possess the ability to act as buffers(i.e., so called “self-buffering”) and therefore may not require theaddition of an exogenous buffer to maintain stable pH (Gokarn et al.,“Self-buffering antibody formulations,” J Pharm Sci. 97(8):3051-66(2008)). Examples of commonly used buffers are listed in Table 2. For amore complete discussion of buffers in biological solutions, see IrwinH. Segel, Biochemical Calculations (2^(nd) ed. 1976), or Remington, TheScience and Practice of Pharmacy 244 (Paul Beringer et al. eds., 21^(st)ed. 2006).

TABLE 2 Buffering Agent pKa Useful pH Histidine 1.82, 6.0, 9.17 5.5-7.4Citrate 3.13, 4.76, 6.40 2.1-7.4 Glycine 2.35, 9.78 2.2-3.6, 8.8-10.6Acetate 4.8 3.8-5.8 Phosphate 7.2 6.2-8.2 Succinate 4.21, 5.64 3.2-6.5Tris 8.06 7.5-9.0 HEPES 7.48 6.8-8.2 MOPS 7.20 6.5-7.9 PIPES 6.766.1-7.5

The term “thermal stabilizer” means an excipient or other additiveincluded in a biopharmaceutical formulation to provide protection to theprotein against thermal degradation, denaturation, and erosion ofbiological activity. Generally, a thermal stabilizer helps maintain theprotein (e.g., antibody) in its native conformation and preventaggregation under conditions of thermal stress. Thermal stress may occurfrom freeze-thaw cycling, exposure to high temperatures, or extensivestorage time. Thermal stabilizers include sugars and othercarbohydrates, sugar alcohols and polyols like polyethylene glycol, andamino acids like glycine. Examples of sugars or sugar alcohols useful asa thermal stabilizer include sucrose, trehalose and mannitol.

The term “hydrophobic interaction media” means a combination of asupport structure and a hydrophobic moiety, wherein the hydrophobicmoiety is affixed to the support structure. The media can be in the formof chromatography media, e.g., beads or other particles held in a packedbed column format, in the form of a membrane, or in any format that canaccommodate a liquid comprising a protein of interest and contaminants.Thus, support structures include agarose beads (e.g., sepharose), silicabeads, cellulosic membranes, cellulosic beads, hydrophilic polymerbeads, and the like. The hydrophobic moiety is the business end of themedia, which binds to hydrophobic molecules and hydrophobic surfaces ofproteins. The degree of hydrophobicity of the media can be controlled byselecting the hydrophobic moiety. For example, the following moietiescan be affixed to media substrate to produce hydrophobic interactionmedia of increasing hydrophobicity, i.e., from low hydrophobicity tohigh hydrophobicity: ether, butyl, octyl, and phenyl. Alkyl groups maybe straight chains or branched. For a review of hydrophobic interactionchromatography and media, see Kuczewski et al., “Development of apolishing step using a hydrophobic interaction membrane adsorber with aPER.C6®-derived recombinant antibody,” Biotech. Bioeng. 105(2):296-305(2010); Roettger and Ladisch, “Hydrophobic interaction chromatography,”Biotechnol Adv. 7(1):15-29 (1989); Shukla and Sanchayita, “Process forpurifying proteins in a hydrophobic interaction chromatographyflow-through fraction,” U.S. Pat. No. 7,427,659 B2, Sep. 23, 2008; andMüller and Franzreb, “Suitability of commercial hydrophobic interactionsorbents for temperature-controlled protein liquid chromatography underlow salt conditions,” J. Chroma. A 1260:88-96 (2012).

Hydrophobic interaction media is employed in a process known ashydrophobic interaction chromatography and is used to separate proteinsof interest from product and process related contaminants. When theprotein of interest is manufactured in and/or purified from host cells,the product and process related contaminants are referred to as hostcell proteins (HCP). HCPs from Chinese hamster ovary (CHO) cells, acommon biotherapeutic manufacturing host cell, can be referred to asCHOPs (Chinese hamster ovary proteins). In some cases, a mixturecontaining the protein of interest (POI) and HCPs are applied to the HICmedia in a buffer designed to promote binding of hydrophobic groups inthe POI to the hydrophobic moiety of the HIC medium. The POI sticks tothe HIC media by binding the hydrophobic moiety, and some HCPs fail tobind and come out in the wash buffer. The POI is then eluted using abuffer that promotes dissociation of the POI from the HIC hydrophobicmoiety, thereby separating the POI from unwanted HCPs.

In some cases, the HIC hydrophobic moiety preferentially binds somecontaminants such as HCPs, and the POI is collected from the HICflow-through. Here, the applicants employ HIC in a flow-through mode,wherein a population of contaminant HCPs, including an esteraseactivity, remain bound to the hydrophobic interaction media.

In some cases affinity chromatography designed to bind specific proteinshaving lipophilic attributes may be employed in lieu of or in concertwith HIC. Since some esterases, such as lipases in general, orphospholipases in particular, bind to triglycerides or phospholipids,molecules that mimic those lipids may be used to capture esterases. Forexample, “myristoylated ADP ribosylating factor 1” (a.k.a. “myrARF1”)can be used to capture a lipase and allow the POI to remain unbound andflow through. To prepare a myrARF1 affinity column, myrARF1 may be boundto Q-sepharose via N-hydroxysuccinimide activation (see Morgan et al.,“Identification of phospholipase B from Dictyostelium discoideum revealsa new lipase family present in mammals, flies and nematodes, but notyeast,” Biochem. J. 382: 441-449 (2004)).

As used herein, the term “container” is meant to include a syringe (asin a pre-filled syringe), a vial (for example a 2.5 mL glass vial forstoring a biopharmaceutical formulation), or any vessel or means tocontain a solid, liquid or gaseous substance. Here, the term “container”is used to refer inter alia to the vessel containing a biopharmaceuticalformulation, as that term is used by the FDA and USP in its guidance onlimitations for subvisible particles (United States Pharmacopeia andNational Formulary (USP 33-NF 28), <788> Particulate Matter inInjections).

EXAMPLES Example 1 Determination of Subvisible Particles in ProteinSamples

The FDA requirement for subvisible particulates in parenteral drugproduct is ≦6,000 particles per container for particles ≧10 micrometersin diameter, and ≦600 particles per container for particles ≧25micrometers in diameter. Presently, no specification exists forparticles of less than 10 micrometers in diameter, but the FDA hasrequested that particles of 2 to 10 micrometers be measured.

Particles of greater than 1 micrometer in diameter were measured usingHIAC light obscuration and Brightwell micro-flow imaging (MFI). HIACcombines light obscuration with laser light scattering enabling thedetection and counting of particles ranging from 500 nm-350 μm in amoving fluid stream. Particles were sized based on voltage responsegenerated in the detector and sorted into pre-determined size rangesbased on voltage response.

For HIAC assays, samples from a manufacturing line (GMP lots) containinga monoclonal antibody at 150 mg/mL were pooled to a total volume of 25mL. For each pooled sample, three readings of five milliliters persample were made. Laboratory samples of the same 150 mg/mL antibodyformulation were also examined by HIAC. Samples from at least threevials (2.5 mL/vial), seven 1-mL syringes (1.14 mL/syringe), or five2.25-mL syringes (2 mL/syringe) were pooled, and three reading of onemilliliter per reading were made. HIAC 9703 and HIAC 8000A instruments(Hach Company, Loveland, Colo.) using the HRLD 400 probe (which reads upto 18,000 cumulative counts per mL) and MC05 probe (which reads up to10,000 cumulative counts per mL) respectively, were used to make thelight obscuration readings.

The MFI method used less material (i.e., 1 mL of formulation, or 1stability vial or syringe) than HIAC light obscuration and yieldedhigher particulate numbers than HIAC. Since MFI is microscopy-based,that method was more sensitive to the translucent protein particulatesand was able to differentiate silicone oil droplets/air bubbles fromprotein particulates for prefilled syringe samples. MFI was conducted ona laboratory sample containing 150 mg/mL of a monoclonal antibody (as inthe HIAC analyses). For MFI, one reading of one milliliter per readingwas made.

Example 2 Failure of Particulate Specification

Two GMP lots of the 150 mg/mL antibody formulation were assessed forsubvisible particles via HIAC light obscuration after at least sixmonths storage at 5° C. The formulation comprised 0.02% polysorbate 20from supplier A, and 150 mg/mL antibody. The antibody was purified fromCHO cell culture using a combination of affinity capture and ionexchange chromatography. No HIC was used. The results are presented inTable 3.

TABLE 3 TIME POINT (months)/ Particle CONDITION (5° C.) 25° C. LOT Size0 6 9 12 6 1 ≧10 μm 125 6,299 13,361 29,505 32,744 2 ≧10 μm 18 353 8,02710,797 18,602

Example 3 Quality and Purity of Fatty Acid Ester Affects SVP Formation

The effect of the nature and quality of the non-ionic detergent(polysorbate 20 and polysorbate 80) on subvisible particle formation ina protein formulation was tested by formulating an antibody in either(i) polysorbate 20 from supplier A (PS20-A), (ii) polysorbate 20 fromsuppler B (PS20-B), or (iii) polysorbate 80 (PS80). Table 4 shows HIACSVP (≧10 μm SVPs) data from the formulated drug substance of thefollowing formula: 20 mM histidine (pH 5.9), 12.5 mM acetate, 0.02%non-ionic detergent (polysorbate), 5% sucrose (w/v), 25 mM arginine, and150 mg/mL antibody, stored as 2.5 mL fill in a 5 mL Type 1 borosilicateglass vial with a West S2-F451 4432/50 GRY B2-40 stopper.

Here, formulated drug substance (“mAb1”) containing polysorbate 80showed significantly less SVP formation over time than thoseformulations containing polysorbate 20. Furthermore, formulationscontaining polysorbate 20 from supplier B (PS20-B), which is a highergrade of polysorbate 80, showed less SVP formation than thoseformulations containing polysorbate 20 from supplier A (PS20-A; a lowergrade of polysorbate 20). A comparative analysis of PS20-A and PS20-Bshows that PS20-B has 5-10% more overall esters than PS20-A, and thatPS20-A has more isosorbide laurate ester than does PS20-B (FIG. 1).

TABLE 4 TIME POINT (months)/ NON-IONIC CONDITION (5° C.) 25° C. LOTDETERGENT 0 6 9 12 6 1 PS20-A 125 6,299 13,361 29,505 32,744 2 PS20-A 18353 8,027 10,797 18,602 1 PS20-B 25 175 NA 1,138 4,221 2 PS20-B 8 108 NA1,198 5,682 1 PS80 19 26 NA 22 92

The stability of polysorbate 20 and polysorbate 80 in the 150 mg/mLantibody (mAb1) formulation containing 0.02% non-ionic detergent(polysorbate) prepared without HIC [process 3, see below and Table 8])were compared, under the hypothesis that degradation of the fatty acidester promotes protein instability and consequent SVP formation. Therelative amounts of remaining esters (mono- and di-esters) weredetermined by mass spectroscopy. Significant degradation of the estercomponents of polysorbate 20 was observed after the samples were storedat 5° C. for six months or 45° C. for two months. Less extensivedegradation was observed for polysorbate 80 under the same conditions(see Table 5). These results correlate with the SVP particle formationobservations.

The rates of degradation of polysorbate 20 and polysorbate 80 formulatedwith 150 mg/mL antibody (mAb1) (as described above for Table 5) weredetermined under identical conditions using mass spectroscopy to measurerelative amounts of free fatty acids and fatty acid esters. Percentester degradation was determined using the following formula:

$\begin{matrix}{{\% \mspace{14mu} {Ester}\mspace{14mu} {Degradation}} = \frac{{\% \mspace{11mu} {POE}\mspace{14mu} {esters}\mspace{14mu} {at}\mspace{11mu} {T0}} - {\% \mspace{11mu} {POE}\mspace{14mu} {esters}\mspace{14mu} {at}\mspace{14mu} {T1}}}{{\% \mspace{11mu} {POE}\mspace{14mu} {esters}\mspace{14mu} {at}\mspace{14mu} T\; 0},}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

wherein T0=time zero, T1=time at experimental condition (i.e., 2 monthsat 45° C.; 6 months at 5° C.), and POE=polyoxyethylene. Table 6 showspercent degradation of polysorbate 20 and polysorbate 80 in 150 mg/mLantibody formulations. The degradation rate of polysorbate 80 wasconsistently lower for mAb1 (but not for all antibodies tested) than thedegradation rate of polysorbate 20 in otherwise identical antibodyformulations. (MAb3 formulations manufactured without HIC showed greaterPS80 degradation than PS20 degradation.)

TABLE 5 Detergent/Chemical Conditions (percent remaining esters) entityTime zero 6 months at 5° C. 2 months at 45° C. PS20 Monoester 100% 60%30% Diester 100% 30% 10% PS80 Monoester 100% 80% 35% Diester 100% 75%25%

TABLE 6 Percent Ester Degradation 6 months at 2 months atPolyoxyethylene ester Time zero 5° C. 45° C. Polysorbate 20 0% 22% 63%Polysorbate 80 0% 13% 47%

Example 4 Polysorbate 20 Degradation Activity

To determine the etiological agent responsible for polysorbate 20degradation, the buffered antibody (150 mg/mL) was separated into twofractions by 10 kDa filtration: a protein fraction, and a bufferfraction. These two fractions, as well as intact buffered antibody, werespiked with 0.2% (w/v) of super refined polysorbate 20 (PS20-B) andstressed at 45° C. for up to 14 days. The study showed (Table 7, part A,columns 1-2) that the protein fraction, not the buffer fraction, had aneffect on the degradation of sorbitan laurate (i.e., the major componentof polysorbate 20), and that the degradation of polysorbate 20 wascorrelated with the concentration of the antibody (Table 7, part B,columns 3-4).

TABLE 7 part A part B % ester Antibody remaining (14 concentration %ester remaining Fraction days at 45° C.) (mg/mL) (12 days at 45° C.)Drug substance 75% 150 82% Protein Fraction 75% 75 92% Buffer Fraction100% 25 98%

Example 5 Hydrophobic Interaction Chromatography

Antibody was produced in a CHO cell host and purified using one of twoprocesses (see Table 8). In one case, the antibody was purified usingion exchangers as polishing steps (capture step, ion exchange 1, ionexchange 2; “Process 3”). In the other case, one of the polishing stepsused to purify the antibody was hydrophobic interaction chromatographyas an additional polishing step (capture step, ion exchange, hydrophobicinteraction; “Process 6”). The antibody, purified by either process 3 orprocess 6, was formulated at 150 mg/mL in 20 mM histidine (pH 5.9), 12.5mM acetate, 5% sucrose, 25 mM arginine, and 0.02% polysorbate 20, andsubjected to forced degradation at 45° C. for up to 14 days. At day 14,about 98% of the sorbitan laurate (i.e., intact ester) remained in theformulation containing the antibody purified using process 6, whereasonly about 28% of the sorbitan laurate remained in the formulationcontaining the antibody purified using process 3. Therefore, thehydrophobic interaction chromatography (HIC) step likely removed anactivity contributing to polysorbate degradation.

TABLE 8 Process Percent Intact No. Purification Steps Polysorbate 20 1Protein A affinity capture (PA) 54% 2 PA > cation exchange (CEX) 25% 3PA > CEX > anion exchange (AEX) 86% 4 PA > CEX > hydrophobic interaction(HIC) 90% 5 PA > AEX 83% 6 PA > AEX > HIC 92%

The role of bulk process steps in removing the putative polysorbatedegradation factor (putative esterase activity) was evaluated. Antibodyproduced from CHO cells was subjected to sequential purification steps,and the stability of polysorbate 20 was assessed at each step. Theresults from one set of experiments are presented in Table 8, whichreports on the percent intact polysorbate 20 at each step or sequence ofsteps. Percent intact polysorbate 20 is predicted to be inverselyproportional to the amount of contaminant esterase activity.

Multiple different antibodies were tested for an associated polysorbatedegrading activity (esterase) and the effect of HIC on that activity. Ineach case, polysorbate 20 degradation activity was detected, and thatactivity was virtually ablated by the incorporation of a HICpurification step (Table 9).

TABLE 9 Antibody HIC or no HIC PS20 Degradation at Day 15 mAb1 (IgG4) NoHIC  72% mAb1 (IgG4) HIC 2.0% mAb2 (IgG1) No HIC  41% mAb2 (IgG1) HIC2.0% mAb3 (IgG4) No HIC  40% mAb3 (IgG4) HIC 5.0% mAb4 (IgG4) No HIC NDmAb4 (IgG4) HIC 1.0%

TABLE 10 Purification Time Point (months)/condition (5° C.) 25° C.Process Surfactant 0 6 12 18 24 36 6 HIC used PS20-A 129 189 140 NR 178NR 136 No HIC PS20-A 35 769 1,342 NR 14,346 NR 1,951 No HIC PS20-B 125401 297 224 NA NA 1,050

The role of HIC in subvisible particle formation was explored. Withoutmeaning to be limited by theory, we hypothesized that the stability ofthe non-ionic detergent in a protein (e.g., antibody) formulation isdirectly correlated to the formation of subvisible particles. Loss ofsurfactant activity may allow protein to aggregate and form subvisibleparticles. Additionally or alternatively, the fatty acids released bythe degrading sorbitan fatty acid esters may also contribute tosubvisible particle formation as immiscible fatty acid droplets.Therefore, levels of subvisible particles ≧10 micrometers in diameterwere counted in drug substance (150 mg/mL antibody in 20 mM histidine(pH 5.9), 12.5 mM acetate, 5% sucrose, 25 mM arginine, and 0.02%polysorbate 20) produced with HIC (e.g., process 6) or without HIC(e.g., process 3). The results (presented in Table 10) show that theapplication of a HIC step significantly reduced the formation of SVPs inthe drug substance (on the order of ten-fold less), even when the lowerquality PS20-A is used.

Example 6 Polysorbate Degradation Assay

Degradation of polysorbate 20 was examined using one or more of severalmethods. The first method employed an enzymatic colorimetric assay toquantify non-esterified fatty acids (NEFA). The NEFA-HR(2) kit (WakoDiagnostics, Richmond, Va.) was used to detect fatty acids in formulateddrug substance containing polysorbate. Briefly, the samples werecombined with ATP and coenzyme A (CoA) in the presence of acyl-CoAsynthetase (ACS). Available (free) fatty acids reacted with the CoA toform acyl-CoA. The acyl-CoA product was reacted with oxygen and acyl-CoAoxidase to produce trans-2,3-dehydroacyl-CoA and hydrogen peroxide.Peroxidase catalyzed the reaction of the hydrogen peroxide with4-aminoantipyrine and 3-methyl-N-ethyl-N-(β-hydroxyethyl)-aniline toform a blue purple pigment (maximum absorbance at 550 nm). The amount ofNEFA in the sample is proportional to the amount of pigment. For adetailed description of the NEFA colorimetric assay, see Duncombe, “TheColorimetric Micro-Determination of Non-Esterified Fatty Acids inPlasma,” Clin Chim Acta. 9:122-5 (1964); Itaya and Ui, “ColorimetricDetermination of Free Fatty Acids in Biological Fluids,” J. Lipid Res.6:16-20 (1965); Novak, M., “Colorimetric Ultramicro Method for theDetermination of Free Fatty Acids,” J. Lipid Res. 6:431-3 (1965); andElphick, M. C., “Modified Colorimetric Ultramicro Method for EstimatingNEFA in Serum,” J. Clin. Pathol. 21(5):567-70 (1968).

The test sample containing the protein of interest (and putative hostcell protein contaminant) was applied to a 10 kDa molecular weightcut-off filter. The retentate was recovered in 10 mM histidine (pH 6.0)at greater than 100 g/L protein and spiked with polysorbate 20 to give atest sample of 100 g/L protein, 0.8% (w/v) polysorbate 20, 10 mMhistidine, pH 6.0 (t_(initial)). The test sample was subjected to 45° C.for 44 hours (t_(final)). Some samples were spiked with oleic acid toevaluate the recovery efficiency of NEFA in the samples. Percentpolysorbate degradation was calculated as follows:

$\begin{matrix}{\frac{{\lbrack{NEFA}\rbrack t_{final}} - {\lbrack{NEFA}\rbrack t_{inital}}}{\lbrack{polysorbate}\rbrack - {\lbrack{NEFA}\rbrack t_{final}}} \times 100\%} & {{Equation}\mspace{14mu} 2}\end{matrix}$

The second method for determining polysorbate degradation was based onmass spectroscopy. Using LC/MS analysis, this assay allowed themeasurement and comparison of the initial percentage of esters andremaining percentage of esters in polysorbates after incubation at 45°C. at different time points. MAb1 produced according to process 6(without HIC and with PS degradation activity) and mAb1 producedaccording to process 3 (with HIC step and without PS degradationactivity) (see Examples 3 and 5, and Table 8) were included as negativeand positive controls, respectively.

Briefly, 15 mg of antibody sample (on the order of 5-10 mg/mL, or 7mg/mL±1.5 mg/mL) was applied to an ultra-filter (Amicon Ultra 50K,Millipore, Billerica, Mass.) and centrifuged at 14,000×g for 15 minutesor until the remaining volume was slightly below the 100 μL marking onthe device. 1 μL of 10% polysorbate was added into the spin filter withthe concentrated protein followed with vortexing. The sample wasrecovered by inverted centrifugation for 5 minutes at 1000 g to recoverthe full volume in the collection tube.

The recovered volume was measured and the concentration of polysorbatecalculated. 1 μL of each recovered sample was and diluted 100-fold in aseparate tube, and the protein concentration measured with Nanodrop 1000(Thermo Fisher Scientific, Inc., Wilmington, Del.). The samples werethen diluted in histidine buffer (10 mM, pH 6.0) and polysorbate stockto achieve 150 mg/mL protein concentration and 0.2% (w/w) polysorbateconcentration.

Time zero (T0) sample (2 μL) was reserved from each sample and stored at−80° C. until used. Samples to be tested were sealed under argon andincubated at 45° C. to induce degradation, and removed for testing atthe prescribed time points. 2 μL was taken from each of the samples ateach time point and diluted with water to 100 μL. Each diluted timepoint sample was stored at −80° C. if storage. After collection of eachtime point, the head space of the sample tube was filled with argon gas,the sample container resealed, and the sample returned to the incubatorto resume incubation.

The time point samples were analyzed using an anion exchange column(Oasis MAX column, 30 μm, 2.1 mm×20 mm; Waters Corporation, Milford,Mass.) followed at t=5 minutes with reverse phase chromatography(ACQUITY UPLC® BEH 130 C4 column, 1.7 μm, 2.1 mm×50 mm; WatersCorporation, Milford, Mass.). The reverse phase output was connected toa mass spectrometer (Thermo Q-Exactive mass spectrometer; Thermo FisherScientific, Inc., Wilmington, Del.). The chromatographic conditions aredescribed in Table 11.

The system was equilibrated with 99% mobile phase A (0.1% formic acid inwater) at a flow rate of 0.1 mL/minute for 40 minutes prior to firstinjection. Water was used as a blank injection. The mass spectrometerparameters were as follows: mass range 150-2000 m/z; heater temperatureat 250° C.; voltage 3.8 kv; sheath gas 40; auxiliary gas 10; capillarytemperature 350° C.; and S-lens 50. When mass spectrometry-basedidentification was not necessary, charged aerosol detection (CAD) wasused an analytical flow rate and a desolvation temperature at 100° C.(Lisa et al., “Quantitation of triacylglycerols from plant oils usingcharged aerosol detection with gradient compensation,” J Chromatogr A.1176(1-2):135-42 (2007); Plante et al., “The use of charged aerosoldetection with HPLC for the measurement of lipids,” Methods Mol Biol.579:469-82 (2009)).

TABLE 11 UPLC System Waters ACQUITY UPLC I-Class/Dionex UltiMate 3000Mobile Phase A: 0.1% formic acid in water B: 0.1% formic acid inacetonitrile Column Waters Oasis ® MAC 30 μm, 2.1 × 20 mm, Part No.186002052 ACQUITY UPLC ® BEH130 C4 column, 1.7 μm, 2 1 mm × 50 mm fromWaters, Part No. 186004496. Column 40° C. ± 1° C. TemperatureAutosampler 5° C. ± 2° C. Temperature Injection Volume 20.0 μL Time Flow(minute) % A % B (μL/minute) Curve Gradient Initial 99.0 1.0 100 Initial1.0 99.0 1.0 100 Linear 5.0 85.0 15.0 100 Linear 40.0 1.0 99.0 100Linear 45.0 1.0 99.0 100 Linear 45.1 99.0 1.0 100 Linear 50.0 99.0 1.0100 Linear

To estimate the total amount of polyoxyethylene (POE), the masschromatogram was extracted using the 300-800 m/z range to avoidinterference from degraded proteins, and the cluster of peaks from about8-15 minutes was integrated. For CAD chromatograms, the first cluster ofPOE peaks was directly integrated from about 8-15 minutes (again,retention time may shift slightly). When there were other speciesco-eluting with the POE, the baseline was adjusted to minimize theirimpact on the peak area.

To estimate the total amount of POE esters, the mass chromatogram wasextracted using the 300-2000 m/z range, and the cluster of peaks fromabout 17-40 minutes was integrated. For the CAD chromatograms, the POEesters peak cluster was directly integrated from about 17-40 minutes.

Percentage of POE esters was calculated according to Equation 3:

$\begin{matrix}{\frac{{POE}\mspace{14mu} {esters}\mspace{14mu} {peak}\mspace{14mu} {area}}{{{POE}\mspace{14mu} {esters}\mspace{14mu} {peak}\mspace{14mu} {area}} + {{POE}\mspace{14mu} {area}}} \times 100\%} & {{Equation}\mspace{14mu} 3}\end{matrix}$

Percentage of remaining POE esters was calculated according to Equation4:

$\begin{matrix}{\frac{\% \; {POE}\mspace{14mu} {esters}\mspace{11mu} {at}\mspace{14mu} {tn}}{\% \; {POE}\mspace{14mu} {esters}\mspace{14mu} {at}\mspace{14mu} {t0}},} & {{Equation}\mspace{14mu} 4}\end{matrix}$

wherein n=2, 4, or 10 days

Example 7 Lipase and Lipase Inhibitors

Polysorbate degradation activity was followed during HIC purification ofan exemplar antibody produced in CHO cell culture. Partially purifiedCHO cell extract was applied to HIC (phenyl-sepharose). Theflow-through, which contained almost all of the antibody, was collectedand analyzed for polysorbate degradation activity. No polysorbatedegradation activity was observed in this flow-through fraction. The HICbound fraction was stripped from the HIC media and subsequentlysubjected to 100 kDa cut-off ultrafiltration/diafiltration. Theunfiltered stripped fraction contained 9.9% polysorbate degradationactivity, the filter permeate contained 1.3% polysorbate degradationactivity and 5% antibody yield, and the filter retentate contained 7.4%polysorbate degradation activity and 95% antibody yield.

TABLE 12 Percent Reduction in Polysorbate 20 Degradation (concentrationof lipase inhibitor) 0 mM Lipase Inhibitor (control) 0.001 mM 0.01 mM0.1 mM Orlistat 0% 0% 27.8%   67% Diethylumbelliferyl 0% 48% 100%  >95%phosphate URB602 0% 0% 20% 0% 2-Butoxyphenyl boronic 0% 0% 28% 0% acid

Whether the polysorbate degrading activity is a lipase was tested bycombining a lipase inhibitor with the polysorbate degrading activityfraction spiked with polysorbate 20. Table 12 presents the data showinga reduction of polysorbate degrading activity due to lipase inhibitorrelative to the control (antibody with associated polysorbate degradingactivity plus polysorbate 20 without lipase inhibitor). Lipaseinhibitors reduced or eliminated the polysorbate degradation activityassociated with the antibody.

Example 8 Putative Phospholipase B-Like 2 Activity

A CHO-produced recombinant antibody HIC strip fraction (not theflow-through), which contained the polysorbate degradation activity, wassubjected to additional HIC in bind/elution mode, wherein the antibodywas eluted with a shallow gradient. Elution fractions were tested forPS20 degradation activity and those fractions having that activity weresubjected to (i) intact mass spec, (ii) native size exclusionchromatography UV analysis (SEC-UV), and (iii) tryptic digestionfollowed with LC-MS and proteomic search analysis. Intact mass spec ofreverse phase liquid chromatography fractions revealed an unknownspecies in hydrophobic fraction L8 (the most hydrophobic fraction).Formulated antibody samples containing polysorbate 20 and spiked with L8(1:100) showed 20% polysorbate degradation by day eight. Antibodymonomer and free light chain were detected in less hydrophobic fractionsL3-L7, as well as L8. Antibody dimer was detected in fractions L5-L8.

HIC strip fractions L3-L9 were subjected to SEC-UV under nativeconditions. Fraction L8 separated into three major peaks coming offfirst, and two minor peaks coming off later and representing smallerspecies. The first peak off the column contained antibody dimer andother oligomers. The second peak contained antibody monomer. The thirdpeak contained the species having polysorbate degradation activity.Thus, the degradation activity is separable from the antibody and is ofsmaller molecular rotation than the antibody monomer.

HIC fraction L8 was also subjected to shotgun proteomics analysis.Briefly, the L8 fraction was sequentially (i) retained on a 10 kDafilter, (ii) reconstituted in 6M guanidine-HCl, 100 mM Tris-HCl, pH 7.5,(iii) treated for 30 minutes at 50° C. in 10 mMTris(2-carboxythyl)phosphine hydrochloride) (TCEP) followed by 30minutes in the dark at room temperature in 20 mM indole-3-acetic acid(IAA), (iv) diluted eight-fold and adding trypsin at 1 part trypsin to20 parts sample and incubated at 37° C. for four hours, and then (v)subjected to LC-MS/MS analysis. Proteomic searching of the resultantpeptide sequences revealed five proteins associated with L8: (i)putative phospholipase B-like 2 (representing 15% of the peak fraction),(ii) peroxiredoxin-1, (iii) heat shock 27 kDa protein 1, (iv)anaphase-promoting complex subunit 1, and (v) U3 small ribonucleoproteinprotein MPP10. joseph

The amount of polysorbate degradation activity correlated with theabundance of phospholipase B-like 2 protein (PLBL2) present. At variouspurification steps, the amount of PLBL2 was determined via nanoLC-MS orLC-MS and the rate of polysorbate degradation (PS20 spiked fractions)was determined. The abundance of PLBL2 was calculated based on the ratioof peptide intensity from the lipase and drug substance (i.e.,antibody). The results are presented in FIG. 2 and Table 13.

TABLE 13 Percent Relative Amount Fraction PS20 Degradation PLBL2 (ppm)²ProA pool 69.02% 991 CEX pool 55.24% 403 AEX pool 12.85% 84 HIC pool8.67% 0 HIC pool 2 10.96% 0 HIC strip 83.75% 1384 mAb1 process 3(example 5) 4.60% 0 mAb1 process 6 (example 29.12% 92 5)¹ ¹Degradationrate adjusted by concentration. ²Abundance of phopholipase calculatedbased on the ratio of peptide intensity from the lipase and drugsubstance.

1. A composition comprising (a) a protein, (b) an intact fatty acidester, and (c) (i) no more than 400 subvisible particles of 10 micronsor greater in diameter, or (ii) no more than 10,000 subvisible particlesof 2 microns or greater in diameter.
 2. The composition of claim 1,wherein the protein is an antibody.
 3. The composition of claim 2,wherein the antibody is at a concentration of at least 30 mg/mL, atleast 40 mg/mL, at least 80 mg/mL, or at least 100 mg/mL.
 4. Thecomposition of claim 1, wherein the fatty acid ester is apolyoxyethylene sorbitan or iso-sorbide fatty acid mono or di-ester. 5.The composition of claim 4, wherein the fatty acid ester is apolyoxyethylene (20) sorbitan laurate.
 6. The composition of claim 1,wherein the composition is free of detectable esterase activity.
 7. Thecomposition of claim 6, wherein the esterase is a carboxylic esterhydrolase (EC 3.1.1).
 8. The composition of claim 7, wherein thecarboxylic ester hydrolase is phospholipase B-like 2 (PLBL2).
 9. Thecomposition of claim 1 comprising a buffer.
 10. The composition of claim1 comprising a thermal stabilizer.
 11. The composition of claim 10comprising a buffer.
 12. The composition of claim 1, wherein thecomposition was stored at 5° C. for at least 6 months.
 13. Thecomposition of claim 12, wherein the molar ratio of fatty acid ester tofree fatty acid is greater than 4 to
 1. 14. The composition of claim 13,wherein the molar ratio of fatty acid ester to free fatty acid is ≧17:3,≧9:1, or ≧19:1.
 15. A process for manufacturing a stable proteinformulation comprising the step of filling a container with a formulateddrug substance, which is essentially free of esterase activity and whichcontains a protein and a fatty acid ester, wherein the formulated drugsubstance is essentially free of subvisible particles.
 16. The processof claim 15, wherein the formulated drug substance is obtained bycontacting a composition, which contains the protein and an esterase,with a hydrophobic interaction medium, and collecting the protein. 17.The process of claim 16, wherein the hydrophobic interaction medium is ahydrophobic interaction chromatography (HIC) resin or an HIC membrane,either of which comprising a hydrophobic moiety affixed to a substrate.18. The process of claim 17, wherein the hydrophobic moiety is selectedfrom the group consisting of a phenyl, ether, butyl, octyl, straightchain alkane, branch chain alkane, 8-carbon alkane, and 18-carbonalkane.
 19. The process of claim 16 comprising the step of concentratingthe protein after the protein is collected from the hydrophobicinteraction medium.
 20. The process of claim 19 comprising the step ofcombining a fatty acid ester with the concentrated protein.
 21. Theprocess of claim 20 comprising the step of combining a buffer and athermal stabilizer with the concentrated protein.
 22. The process ofclaim 15, wherein the composition is stored at 5° C. for at least 6months.
 23. The process of claim 15, wherein the container contains lessthan 400 particles having a diameter of ≧10 microns.
 24. The process ofclaim 15, wherein the container contains less than 10,000 particleshaving a diameter of ≧2 microns.
 25. The process of claim 15, whereinthe protein is an antibody.
 26. The process of claim 25, wherein theantibody is a recombinant human monoclonal antibody.
 27. The process ofclaim 15, wherein the esterase is a carboxylic ester hydrolase.
 28. Theprocess of claim 27, wherein the carboxylic ester hydrolase is aphospholipase B-like
 2. 29. The process of claim 28, wherein thephospholipase B-like 2 comprises the amino acid sequence of SEQ ID NO:1.30. The process of claim 15, wherein the fatty acid ester is apolyoxyethylene (20) sorbitan ester.
 31. The process of claim 30,wherein the polyoxyethylene (20) sorbitan ester is polysorbate 20 orpolysorbate 80.